Project Summary/Abstract Brain-derived neurotrophic factor (Bdnf) is a well-studied gene that is known to play a critical role in brain development, neuronal differentiation, survival, and plasticity. Rodent Bdnf consists of nine 5? non-coding exons (I-IXa) and one 3? coding exon (IX). Each non-coding exon has its own unique upstream promoter region, where transcription can be initiated. Through differential splicing, several Bdnf mRNA transcript variants are produced, which all code for the same mature Bdnf protein. The complex structure of the Bdnf gene allows for tight spatiotemporal regulation of its specific variant expression, guided by environmental stimuli. The regulation of Bdnf transcription has been extensively studied, however, no clear mechanism of differential variant induction has been identified and it is still unclear why so many transcripts exist to encode one single protein. The overall goal of this proposal is to understand the activity-dependent regulation of different Bdnf transcript variants and the role of specific transcripts in guiding cellular physiology and animal behavior. CRISPR/dCas9 fusion constructs will be used to selectively manipulate the expression of one Bdnf transcript variant at a time in hippocampal cultured neurons. Fused to the catalytically inactive dCas9, effector proteins that promote transcriptional activation, VP64, or repression, KRAB, will be targeted to a specific Bdnf promoter using a short guide RNA (gRNA) to modify epigenetic profiles and alter gene expression. The induction of all Bdnf variants and other plasticity-regulated genes will be analyzed using qRT-PCR and whole-genome RNA sequencing. Furthermore, since Bdnf expression is essential for synaptic plasticity, the induction of long-term potentiation (LTP) in brain slices, as well as new memory formation in live animals, CRISPR/dCas9 tools will be used to target specific Bdnf transcript variants in vivo. By selectively upregulating a single Bdnf splice variant, the functional role of individual Bdnf transcripts will be tested for the induction of LTP or contextual memory formation. This study will shine light on the regulation of Bdnf transcript variant expression in neuronal plasticity and assess the therapeutic potential of site-specific epigenome editing of the Bdnf gene in synaptic plasticity disorders using the cutting-edge CRISPR/dCas9 tools.